Betaine-homocysteine S-methyltransferase deficiency causes increased susceptibility to noise-induced hearing loss associated with plasma hyperhomocysteinemia.

Betaine-homocysteine S-methyltransferases (BHMTs) are methionine cycle enzymes that remethylate homocysteine; hence, their malfunction leads to hyperhomocysteinemia. Epidemiologic and experimental studies have revealed a correlation between hyperhomocysteinemia and hearing loss. Here, we have studied the expression of methionine cycle genes in the mouse cochlea and the impact of knocking out the Bhmt gene in the auditory receptor. We evaluated age-related changes in mouse hearing by recording auditory brainstem responses before and following exposure to noise. Also, we measured cochlear cytoarchitecture, gene expression by RNA-arrays and quantitative RT-PCR, and metabolite levels in liver and plasma by HPLC. Our results indicate that there is an age-dependent strain-specific expression of methionine cycle genes in the mouse cochlea and a further regulation during the response to noise damage. Loss of Bhmt did not cause an evident impact in the hearing acuity of young mice, but it produced higher threshold shifts and poorer recovery following noise challenge. Hearing loss was associated with increased cochlear injury, outer hair cell loss, altered expression of cochlear methionine cycle genes, and hyperhomocysteinemia. Our results suggest that BHMT plays a central role in the homeostasis of cochlear methionine metabolism and that Bhmt2 up-regulation could carry out a compensatory role in cochlear protection against noise injury in the absence of BHMT.-Partearroyo, T., Murillo-Cuesta, S., Vallecillo, N., Bermúdez-Muñoz, J. M., Rodríguez-de la Rosa, L., Mandruzzato, G., Celaya, A. M., Zeisel, S. H., Pajares, M. A., Varela-Moreiras, G., Varela-Nieto, I. Betaine-homocysteine S-methyltransferase deficiency causes increased susceptibility to noise-induced hearing loss associated with plasma hyperhomocysteinemia.

Human betaine-homocysteine methyltransferase (BHMT) and BHMT2: common gene sequence variation and functional characterization.

Betaine-homocysteine methyltransferase (BHMT) catalyzes the remethylation of homocysteine. BHMT2 encodes a protein 73% identical in amino acid sequence to BHMT, but the function of BHMT2 remains unclear. We set out to identify and functionally characterize common genetic variation in BHMT and BHMT2. Specifically, we sequenced exons, exon-intron splice junctions and the 5'-flanking regions (5'-FRs) of BHMT and BHMT2 using 240 DNA samples from four ethnic groups. Twenty-five single nucleotide polymorphisms (SNPs), including 4 nonsynonymous SNPs, and 39 SNPs, including 4 nonsynonymous, were observed in BHMT and BHMT2, respectively. BHMT wild type (WT) and variant allozymes were expressed in COS-1 cells. Variant allozymes showed no significant differences from WT in levels of enzyme activity or immunoreactive protein, but there were statistically significant differences in apparent K(m) values. Luciferase reporter gene constructs were created for the three most common BHMT 5'-FR haplotypes, and significant variation was observed in the ability of these constructs to drive transcription. Although BHMT2 mRNA has been observed in human liver and kidney, expression of the protein has not been reported. We were unable to express BHMT2 in mammalian cells, and the protein aggregated after bacterial expression. Furthermore, BHMT2 was rapidly degraded in a rabbit reticulocyte lysate, but it could be stabilized by cotransfection of COS-1 cells with BHMT and, after cotransfection, it coprecipitated with BHMT. These studies have defined common genetic variation in BHMT and BHMT2 and functionally characterized BHMT SNPs. They may also help to explain why BHMT2 has not previously been defined functionally.

Effect of transgenic extrahepatic expression of betaine-homocysteine methyltransferase on alcohol or homocysteine-induced fatty liver.

BACKGROUND: Chronic alcohol feeding induces hyperhomocysteinemia (HHcy). Previously, we reported a protective role of betaine-homocysteine methyltransferase (BHMT) in homocysteine-induced injury in cultured hepatocytes. In this study, we investigated the direct role of BHMT in alcohol or homocysteine-induced liver injury. METHODS: Betaine-homocysteine methyltransferase transgenic (Tg) mice were generated. Comparisons were made between the Tg and wild type (WT) mice in their response to intragastric alcohol infusion or to oral feeding of a high methionine low folate diet (HMLF). RESULTS: Expression of the Tg BHMT was increased in organs peripheral to the liver. The alcohol infusion for 4 weeks increased: plasma ALT by 5-fold in WT mice and 2.7-fold in Tg mice; plasma homocysteine by 7-fold in WT mice and 2-fold in Tg mice; liver triglycerides by 4-fold in WT mice and 2.5-fold in Tg mice. The alcohol-induced fatty liver was more severe in WT than in Tg mice based on H&E staining. The HMLF feeding for 4 weeks increased plasma ALT by 2-fold in WT mice and 1-fold in Tg mice; plasma homocysteine by 21-fold in WT mice and 3.3-fold in Tg mice; liver triglycerides by 2.5-fold in WT mice and 1.5-fold in Tg mice. HMLF induced accumulation of macro fat droplets in WT but not Tg mice. Betaine supplementation decreased partially the alcohol or HMLF-induced increase of ALT, homocysteine and liver lipids in WT mice. However, Tg mice were normal when fed both HMLF and betaine. In WT mice, both alcohol and HMLF induced moderate increase of sterol regulatory element binding protein 1 (SREBP1) protein which was partially reduced by betaine supplementation. In Tg mice, alcohol but not HMLF increased SREBP1. Carbohydrate responsive element-binding protein was increased by alcohol in either WT or Tg mice which was not affected by betaine supplementation. Ratio of S-adenosylmethionine (SAM) to S-adenosylhomocysteine (SAH) was reduced by 50% in WT and by 20% in Tg mice fed alcohol. Ratio of phosphatidylcholine (PC) to phosphatidylethanolamine (PE) was reduced in WT but not Tg mice fed alcohol. Changes in PE methyltransferase activities were not detected in response to alcohol or HMLF feeding but were increased by betaine. CONCLUSIONS: The BHMT Tg mice are resistant to alcohol or HMLF-induced HHcy and liver steatosis indicating that peripheral metabolism of homocysteine protected the liver without a direct effect of BHMT in the liver. Multiple mechanisms are involved in protection by betaine including increased SAM/SAH and PC/PE ratios.

[Homocysteine metabolism].

Homocysteine, a sulfur amino acid, is an intermediate metabolite of methionine. In 1969, McCully reported autopsy evidence of extensive arterial thrombosis and atherosclerosis in children with elevated plasma homocysteine concentrations and homocystinuria. On the basis of this observation, he proposed that elevated plasma homocysteine (hyperhomocysteinemia) can cause atherosclerotic vascular disease. Hyperhomocysteinemia is now well established as an independent risk factor for atherosclerotic vascular disease. Mild hyperhomocysteinemia is quite prevalent in the general population. It can be caused by genetic defects in the enzymes involved in homocysteine metabolism or nutritional deficiencies in vitamin cofactors, certain medications or renal disease. An increase of 5 micromol per liter in the plasma homocysteine concentration raises the risk of coronary artery disease by as much as an increase of 20 mg per deciliter in the cholesterol concentration. In this article, we review the biochemical, experimental and clinical studies on hyperhomocysteinemia, with emphasis on the metabolism and pharmacokinetics of homocysteine.

Depletion of Bhmt elevates sonic hedgehog transcript level and increases ß-cell number in zebrafish.

Betaine homocysteine S-methyltransferase (BHMT, EC 2.1.1.5) is a key enzyme in the methionine cycle and is highly expressed in the liver. Despite its important biochemical function, it is not known whether BHMT plays a role during organ development. In this report, we showed that early in development of zebrafish before endoderm organogenesis, bhmt is first expressed in the yolk syncytial layer and then after liver formation becomes a liver-enriched gene. By using the anti-bhmt morpholinos that deplete the Bhmt, we found that in morphant embryos, several endoderm-derived organs, including liver, exocrine pancreas, and intestine are hypoplastic. Strikingly, the number of ß-cells in the pancreatic islet was increased rather than reduced in the morphant. Additional studies showed that Bhmt depletion elevates the sonic hedgehog (shh) transcript level in the morphant, whereas Bhmt-depletion in the Shh-deficient mutant syu failed to rescue the isletless phenotype. These molecular and genetic data strongly suggest that Shh functions downstream of Bhmt to promote ß-cell development. Therefore, although there are still many intriguing questions to be answered, our finding may identify a novel function for Bhmt involving modulation of Shh signaling to control ß-cell development.

Yeast, plants, worms, and flies use a methyltransferase to metabolize age-damaged (R,S)-AdoMet, but what do mammals do?

The biological methyl donor S-adenosyl-L-methionine [(S,S)-AdoMet] can spontaneously break down under physiological conditions to form the inactive diastereomer (R,S)-AdoMet, which may interfere with cell function. Although several lower organisms metabolize (R,S)-AdoMet via homocysteine methyltransferases, it is unclear how mammals deal with it. In this paper, we show that the mouse liver extracts, containing the BHMT-2 homocysteine methyltransferase candidate for a similar activity, recognizes (S,S)-AdoMet but not (R,S)-AdoMet. We find no evidence for the enzymatic breakdown of (R,S)-AdoMet in these extracts. Thus, mammals may metabolize (R,S)-AdoMet using a different strategy than other organisms.